PsbO, the manganese-stabilizing protein: Analysis of the structure–function relations that provide insights into its role in photosystem II

Journal of Photochemistry and Photobiology B: Biology 104 (2011) 179–190

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Short Review

PsbO, the manganese-stabilizing protein: Analysis of the structure–function relations that provide insights into its role in photosystem II Hana Popelkova a,⇑, Charles F. Yocum a,b a b

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

a r t i c l e

i n f o

Article history: Available online 21 January 2011 Keywords: Structure-function Manganese-stabilizing protein Mutation Natively unfolded polypeptide Photosystem II

a b s t r a c t The minireview presented here summarizes current information on the structure and function of PsbO, the photosystem II (PSII) manganese-stabilizing protein, with an emphasis on the protein’s assembly into PSII, and its function in facilitating rapid turnovers of the oxygen evolving reaction. Two putative mechanisms for functional assembly of PsbO, which behaves as an intrinsically disordered polypeptide in solution, into PSII are proposed. Finally, a model is presented for the role of PsbO in relation to the function of the Mn, Ca2+, and Cl cofactors that are required for water oxidation, as well as for the action of hydroxide and small Mn reductants that inhibit the function of the active site of the oxygen-evolving complex. Ó 2011 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PsbO structure in solution and in the PSII-associated form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. PsbO is a thermostable natively unfolded protein in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Interaction of PsbO with PSII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. PsbO stoichiometry in PSII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. PSII intrinsic subunits/residues that participate in binding of PsbO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Amino acid residues of PsbO participating in its interaction with PSII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Functional assembly of PsbO into PSII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of PsbO in PSII. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. PsbO and the OEC active site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The role of PsbO in relation to the inorganic cofactors in the OEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. PsbO and manganese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. PsbO and Ca2+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. PsbO and Cl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. The effect of PsbO on the S-states and Y
Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179 180 180 181 181 181 182 182 184 184 185 185 185 186 186 187 187 187 187 187

1. Introduction

⇑ Corresponding author. Address: Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109-1048, USA. Tel.: +1 734 764 9543; fax: +1 734 647 0884. E-mail address: [email protected] (H. Popelkova). 1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2011.01.015

The largest extrinsic subunit of the membrane-associated photosynthetic redox enzyme called photosystem II (PSII) is PsbO, a 26.5 kDa polypeptide also called the manganese-stabilizing protein, which is located on the lumen side of thylakoid membranes. The PSII reaction center consists of a core of intrinsic proteins:

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D1 and D2 bind the Chl a, Pheo a, and plastoquinone cofactors that participate in light-catalyzed charge separation; the larger chlorophyll-binding polypeptides CP47 and CP43 and cytochrome b559, along with a number of small protein subunits are also associated with the core structure of the photosystem [1,2]. Three inorganic cofactors (4 Mn, 1 Ca2+, 1 Cl ) and at least three extrinsic proteins (one of them PsbO) are bound to the PSII core proteins, and together they form a module called the oxygen-evolving complex (OEC), which is the catalytic center for water oxidation [3–6]. The OEC is assembled in a stepwise process termed ‘‘photoactivation’’, in which Mn2+ atoms are incorporated (photoligated) into the preassembled inactive apo-S-state complex that is activated by assembly of the Mn cluster [7,8]. Photoligation involves oxidation by light of Mn to states higher than +2 and binding of the metals to sites within the OEC. Calcium is not directly involved in the photoligation reaction, but it does function in Mn photoactivation [9] by preventing inhibitory ligation of the Mn ions in higher redox states (MnP3+) [10]. On the other hand, PsbO is not essential for photoligation of Mn, but is absolutely required for maximal efficiency of the water-oxidation reaction [11]. The dispensability of PsbO in Mn photoligation experiments provides evidence to suggest that PsbO is not required for binding of Mn to PSII and that as such it has no amino acid residues that function as direct ligands to the Mn cluster. While PsbO is unnecessary for binding of a substrate water molecule to the Mn cluster, at least in the S1 state [12], its indispensable role in the oxygen evolution reaction has been demonstrated by the consequences of its extraction from PSII, which can be implemented by washing intact samples with alkaline-Tris [13], 1 M CaCl2 [14] or MgCl2 [15], or 0.2 M NaCl–2.6 M urea [16]. In the latter case, the combination of ionic strength and a chaotropic agent dissociates the protein under fairly mild conditions. In contrast, CaCl2 and MgCl2 at high concentrations disrupt electrostatic interactions between PsbO and PSII. A common feature of these manipulations is that in all cases, except for alkaline-Tris, the Mn cluster is retained in intact form. Tris at high pH exerts multiple effects on Mn and polypeptide binding, since both PsbO and the Mn cluster are released from PSII [17]. Caution is required in the use of CaCl2-treated PSII where the role of Ca2+ is being characterized; residual concentrations of this cofactor can remain in a sample after the treatment [18]. Recently, Yu et al. [19] reported that PsbO can also be removed from spinach PSII by HgCl2 at micromolar and higher concentrations without removing the PsbP and PsbQ extrinsic proteins. The authors hypothesized that Hg could react with the lone disulfide bridge of PsbO and replace it with the –S–Hg–S– motif, which would eventually result in a conformational change in the PsbO structure, and its release from PSII. Additional experiments are needed to confirm this interesting hypothesis. Since isolation of PSII usually employs spinach leaves [20], in vitro depletion of PsbO from such preparations reveals defects that are created in the activity of eukaryotic PSII. Typically, PsbO removal causes a decrease in the rate of O2 evolution to 20% of that observed with intact PSII, and the sample requires high concentrations of Ca2+ and especially Cl that prevents loss of two of four Mn atoms from the OEC [14,16,18]. In eukaryotic organisms, PsbO is encoded by nuclear DNA, and is imported into chloroplasts as a precursor [21]. Mutations that deleted PsbO from PSII were found to have different effects in prokaryotes and eukaryotes. The DPsbO mutant from Synechocystis sp. PCC 6803 assembles PSII, evolves oxygen at low rates, and grows slowly under photoautotrophic conditions, although it is light sensitive [22]. On the other hand, cells Chlamydomonas reinhardtii carrying the DpsbO mutation could not grow photoautotrophically, and did not accumulate PSII [23]. A similar phenotype was observed in Arabidopsis thaliana, where both genes for the PsbO isoforms PsbO-1 and PsbO-2 were suppressed by RNAi [24]. Expression of

only the PsbO-2 protein in the psbo1 A. thaliana mutant caused retarded photoautotrophic growth [25], longer lifetimes of the S2 and S3 states, higher accumulation of PSIIb reaction centers, and retardation of QA ? QB electron transfer [26]. More details on the consequences of in vitro or in vivo removal of PsbO from PSII are presented in Section 3 (function of PsbO in PSII). A prior PsbO minireview focused on its solution structure [27]. Since then, several reviews on PsbO that included analyses of the PsbO structure in solution or in the PSII-associated form were published [28–31]; many of them were based on the crystallographic models of PsbO from thermophilic cyanobacteria [2,32,33]. Here, information on PsbO is reviewed in the context of its behavior as a natively unfolded protein, and aspects of PsbO structure are examined that are most relevant to the protein’s proper assembly and function in PSII. Two scenarios for a mechanism of functional association of PsbO’s flexible loops into PSII are also proposed. With an emphasis on PsbO function in PSII, results of biochemical, spectroscopic, and mutagenesis experiments that provide some insights into the effect of PsbO on the inorganic cofactors, the Sstates, and reduction of Y
Z in the OEC are reviewed. A schematic model summarizing the role of PsbO in relation to the function of the Mn, Ca2+, and Cl cofactors and to the action of hydroxide and small Mn reductants, such as dimethylhydroxylamine, in the OEC active site is presented.

2. PsbO structure in solution and in the PSII-associated form 2.1. PsbO is a thermostable natively unfolded protein in solution In solution, PsbO behaves as a natively unfolded or intrinsically disordered polypeptide [27,34,35]. Proteins that belong to this family (see the Disprot database (http://www.disprot.org)) possess several characteristics in common that cannot be found in normally-folded globular proteins. These properties include: (1) Secondary structure that contains predominantly b-sheet and large amounts of turns and random coils (see [27,30,36,37]). (2) Folding that is not assisted by chaperones, but rather by binding to a supermolecular complex of which the intrinsically disordered protein is a subunit [38,39]. (3) Overestimated molecular size based on SDS–PAGE or on gel filtration [40–43]. (4) Unusually large Stokes (hydrodynamic) radius [40,44]. (5) Extremely acidic or basic pI values (PsbO has pI of 5.2) [40,45]. (6) A high ratio of disorder promoting residues (A, R, G, Q, S, P, D, E, and K) to hydrophobic (W, C, F, I, Y, V, L, and N) residues [39,40,45]. (7) A resistance to heat denaturation that is unusual for mesophilic proteins [40,46–48]. Thermostability of PsbO and other natively unfolded mesophilic proteins could be a leftover from the era when the earth’s atmosphere had a higher temperature compared to the present day, or it could be a consequence of an embedded physical/chemical property of amino acid sequences or overall protein charge. In the case of PsbO, there appears to be a subtle equilibrium between protein’s thermostability and function. It has been shown recently that highly thermostable mutants, where Phe replaces W241 or Y242, exhibit low activity [49] because they cannot undergo overall folding that is important for their functional assembly into PSII [50,51]. However, removal of six N-terminal residues from W241F PsbO can significantly restore function to a W ? F mutant, because it reduces its thermostability to a near-wild-type level [49,52]. Shutova

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et al. in [53] proposed that PsbO thermostability was facilitated by the presence of its lone disulfide bridge. However, Betts et al. [54] had shown that the disulfide bridge was not required for PsbO function, and a later study that employed the C28AC51A PsbO mutant, which eliminates the disulfide bond, showed that it was unnecessary for PsbO’s characteristic thermostability in solution [55]. Instead, examination of the effects of protein concentration and pH on heat-induced aggregation showed that PsbO is thermostable because it resists aggregation more effectively than either bovine serum albumin or carbonic anhydrase, two model globular water soluble proteins that were subjected to the same heat treatments. It is likely that PsbO’s heat stability is a consequence of a high content of charged residues on the surface of the protein that interfere with aggregation by charge repulsion [56]. The ability of PsbO to resist aggregation supports its classification as a natively unfolded polypeptide, and might also be a useful property during its binding and assembly into PSII, where the protein can occur at high concentrations in the thylakoid lumen [57]. The other intrinsically disordered characteristic of PsbO, a high fraction of unordered elements in its secondary structure, likely enables PsbO to undergo structural rearrangements when it interacts with PSII [40]; intrinsically disordered proteins are known to be important elements of multi-subunit complexes [39]. 2.2. Interaction of PsbO with PSII 2.2.1. PsbO stoichiometry in PSII The number of copies of PsbO bound per PSII reaction center is still a matter of debate. The crystal structures of cyanobacterial PSII complexes reveal one copy of PsbO per PSII [2,32,33], as do some early biochemical studies on spinach [58,59]. Difficulties with the techniques used in these studies are discussed in [60,61]. Early low-resolution electron microscopy images have also been interpreted to indicate one subunit of each extrinsic protein per PSII monomer [62,63]. The authors of these studies assumed that PsbO is a globular protein, which is in contrast with reports showing that it exhibits a prolate ellipsoid shape in solution [41,42], and the corresponding b-barrel shape in PSII crystal structures [2,32,33]. Consistent with the elongated shape of PsbO, later results of electron microscope studies have been interpreted to indicate that PsbO has an aglobular shape and/or is present in two copies per PSII [64]. A recent review on supramolecular organization of thylakoid membrane proteins in green plants suggests the possibility that the second PsbO copy might be present in PSII monomers and would not be seen in top-view electron microscope images if it affects staining of PSII [65]. At the present time, much of the experimental evidence from biochemical experiments and from experiments involving site-directed mutagenesis of PsbO favors the presence of two PsbO subunits per higher plant PSII reaction center: (1) Isolation of PsbO from spinach by Yamamoto et al. [66] yielded >1.7 mol of PsbO per mol PSII, which is likely to underestimate the actual value owing to losses of protein during purification. (2) Immunological quantification of PsbO using internal PsbO standards or PSII containing various amount of Mn per PSII reaction center yielded a PsbO content in the range of 2.0– 2.13 mol of PsbO per mol PSII [60,67]. (3) Binding curves for wild-type PsbO and reconstitution curves for some PsbO mutants exhibited some sigmoidicity indicative of cooperative binding, which would require at least two copies of PsbO per PSII monomer [43,68,69]. The binding of the first PsbO subunit increases the affinity for binding of the second PsbO subunit, a phenomenon unattainable with a single copy of PsbO per PSII.

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(4) A differential replacement study showed that the V235A PsbO mutant competes with the wild-type protein for one of the two binding sites and that the mutant binds tightly to only one of two sites [70]. (5) Work on A. thaliana revealed that two PsbO isoforms (PsbO1, PsbO2) bind equally to PsbO-depleted PSII when incubated with one mole of each isoform [71], indicating that there are two PsbO binding sites in PSII. (6) Studies employing PsbO truncation mutants showed that the N-terminus of higher plant PsbO’s contains two sequences that are required for binding of two copies of the subunit to PSII. Cyanobacterial PsbO’s lack one of the two sequences [72], which is consistent with only one PsbO subunit that is found in the present crystal structures of cyanobacterial PSII [2,32,33]. (7) Work focused on inorganic cofactor stabilization and retention revealed that each PsbO copy in PSII from spinach has a unique function in respect to retention and or stabilization of Mn and Cl [73]; the first copy of PsbO stabilizes the Mn cluster and enhances Cl retention, while the second PsbO copy optimizes Cl retention in order to maximize O2-evolution activity [73]. Despite all of the experimental evidence, the question of PsbO stoichiometry in PSII will probably remain a matter of debate until it is unambiguously resolved by high-resolution crystal structures of prokaryotic and eukaryotic PSII’s, and the PsbO stoichiometry in cyanobacteria has been explored in detail in the context of the functions of the other extrinsic subunits that are found in PSII from these organisms [31,74]. A single subunit of PsbO per PSII found in the crystal structures of cyanobacterial PSII [2,32,33] suggests that cyanobacterial and higher plant PSII’s exhibit different PsbO stoichiometries, which is consistent with differences in the N-terminal PSII binding domains of their corresponding PsbO’s [52,72]. It is possible that cyanobacterial PSII (and red algal PSII probably as well [see 52]) posses some other extrinsic subunit, perhaps PsbV, that partially replaces the functional/structural role of the second copy of PsbO [31]. The green algal PsbO sequence contains two PSII binding domains, which would indicate the presence of two PsbO subunits per PSII in green algae [52], but direct biochemical experiments are needed to confirm this assumption. 2.2.2. PSII intrinsic subunits/residues that participate in binding of PsbO In a search for the PSII subunits that interact with PsbO, application of biochemical approaches produced numerous results. Early cross-linking studies indicated that PsbO binds to CP47 [75]; see also [61]. This result has been confirmed and further elaborated by Odom and Bricker [76], who found that the domain 364E–440D (E loop) of CP47 crosslinks to the N-terminal domain 1E–76K of PsbO. Later work by Ohta et al. showed that 87D and/or 94E of the A loop of CP47 also crosslinked to PsbO [77]. A NHS-biotinylation study revealed that the domains 304K–321K and 389K–419K in the large extrinsic E loop of CP47 are biotinylated when PsbO is removed from PSII, indicating that these two regions interact with PsbO [78]. Analysis [see [61]] of the results on mutated cyanobacterial strains obtained by PutnamEvans et al. [79,80] concluded that the 384R and 385R residues in the loop E of CP47 form a binding site for PsbO, and that the carboxyl groups of PsbO likely interact electrostatically with these arginyl residues of CP47 [61]. Gleiter et al. found that a deletion of the region around 373A–380D of CP47 in Synechocystis sp. PCC 6803 causes the phenotype with impaired PsbO binding [81]. Further biochemical experiments revealed that, in addition to CP47, the CP43 polypeptide and the cytochrome b-559 a-subunit also interact with PsbO when it is associated with PSII [82,83]. Enami et al. compared trypsin-digested products and found that the domain 357R–358F in the loop E

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of CP43, the peptide bond between 457K and 458G in CP43, and the Cterminus of the cytochrome b-559 a-subunit were shielded from a tryptic attack when PsbO was bound to PSII, but not in its absence, indicating that these domains/residues are needed for binding of PsbO to PSII [82]. Rosenberg et al. found that Synechocystis sp. PCC 6803 PsbO has a significant binding defect when the mutation E352Q is introduced to the sequence of CP43[83]. Those results that identify domains of PSII intrinsic polypeptides that are shielded by PsbO suggest, but by no means show, that amino acid residues in such domains interact directly with PsbO. It is reasonable to suppose that charged residues within these domains could form electrostatic interactions with complementary residues of PsbO. An alternative approach to biochemical manipulations, which can also reveal the PSII-associated interacting partners of PsbO, is crystal structures. The crystallographic model of higher plant PSII is not currently available, but some insights into the interaction between PsbO and PSII can be obtained from the crystal structure of cyanobacterial PSII (PDB entry 1S5 l [32]; 2AXT [33]; 3BZ2 [2]), in spite of the fact that organization of subunits in cyanobacterial PSII differs somewhat from that in higher plants [1,31]. Although caution is advised in drawing conclusions about PSII structure deduced from the crystallographic models of PSII, due to a radiation damage that reduced the Mn atoms, positions of protein backbones can be assumed to be more or less reliable in many of these models (J. Kern, personal communication); precise assessment of the amino acid side-chains requires further improvement in the resolution of PSII crystal structures. It is interesting that the current structural model for PsbO bound to cyanobacterial PsbO does not show interactions between the N-terminus of this protein and the large extrinsic loop of CP47. Instead, the N-terminus of the single copy of PsbO in these structures is observed to interact with the large extrinsic loop of CP43. The CP47 subunit of cyanobacterial PSII is observed to interact with the large flexible loop of PsbO of the same PSII monomer and with two smaller flexible loops of PsbO of the opposite monomer. The interaction between PsbO and CP47 of the opposite monomers may facilitate stabilization of PSII dimmers in crystals of the cyanobacterial photosystem. In higher plant PSII, interactions between its subunits, including PSII dimerization, will only be understood when a highresolution crystal structure from higher plants becomes available.

2.2.3. Amino acid residues of PsbO participating in its interaction with PSII A number of studies searched for PsbO amino acid residues that are involved in its interaction with PSII (for a review see [30,31]). Cross-linking and modification experiments with EDC, glycine methyl ester, and sulfo-(N-hydroxy)-succinimide showed that both positively and negatively charged amino acid residues participate in PsbO binding to PSII [84–86]. Four domains of PsbO (1E–4K, 20 K, 101K–105K, and 159K–186K) were labeled with NHS-biotin only when PsbO was free in solution and not when it was associated with PSII, indicating that these regions of PsbO interact with PSII [87]. The residue 160K in PsbO from thermophilic Synechococcus elongatus that is homologous to 159K in spinach PsbO was also found to be essential for binding of PsbO to PSII [88]. Results of a mutagenesis study on the D9N and D9K mutants of spinach PsbO were interpreted to indicate that the PsbO N-terminus is not directly involved in binding of the protein to PSII [89], but exposure of PsbO to proteolytic digestion with chymotrypsin or V8 protease revealed that the truncated protein is unable to bind to PSII after removal of 16 or 18 N-terminal amino acids [90]. These conflicting observations were explored further by experiments that employed N-terminal deletion mutations of spinach PsbO, which concluded that Thr7 and Thr15 are required for functional binding of two eukaryotic PsbO subunits to PSII [91]. These Thr residues might form H-bonding interactions with the intrinsic domain of PSII so

Table 1 Amino acid sequence alignment of the domains of natively unfolded polypeptides that are partially homologous to the TYDE or TYLE motifs in PsbO. Natively unfolded proteins shown in this table were found on the DisProt website (http://www.disprot.org/), where they were categorized in the functional subclass ‘‘protein–protein binding’’. DP00XXX denotes DisProt identification code.

as to position the protein for the interactions that allow for high affinity binding of PsbO to PSII. A number of N-terminal PsbO residues that can be removed without affecting specific binding of PsbO to PSII have been identified, but their deletion triggers nonspecific interactions between PsbO and PSII. These residues are part of the N-terminal domains 1 EGGKR6L, 8YDEIQS14K, and 16YL18E that are needed to prevent nonspecific binding of the protein to PSII [43,91]. Thr7 and Thr15 at the N-terminus of spinach PsbO are part of the conserved amino acid motifs TYDE and TYLE [72]. Since both of these motifs are localized in the random coils of PsbO that participate in interaction of PsbO with the PSII intrinsic proteins, other natively unfolded polypeptides were examined to see whether they contain similar motifs. Indeed, a number of intrinsically disordered proteins, which belong to the subfamily of polypeptides that participate in protein–protein interactions, contain similar motifs in their primary amino acid sequence (see Table 1). For most of these proteins, crystal structures are not available. However, for E3 ubiquitin-protein ligase (PDB entry 3IUX [92]) and for glucocorticoid receptor (PDB entry 3E7C [93]), the crystallographic models show that the domains that are partially homologous with the TYDE and TYLE motifs of PsbO are localized in unordered turns (as they are in PsbO), or occupy both unordered structures and a-helical elements. This would indicate that some intrinsically disordered proteins appear to employ sequence motifs similar to those of PsbO when they participate in protein–protein interactions within multi-subunit complexes. 2.2.4. Functional assembly of PsbO into PSII A closer examination of all primary amino acid sequences of PsbO, which are currently available in the protein database (http://www.ncbi.nlm.nih.gov/protein), reveals that the lengths of mature PsbO proteins are more or less the same [52]. Mutations to PsbO that induced changes in the total length of the mature protein were found to have significant consequences for its function. Motoki et al. [88] found that insertion of G, A, or V between positions 156 and 157 or deletion of L157 from the primary sequence of cyanobacterial PsbO yields mutated proteins that are unable to restore activity of the reconstituted PSII complex from thermophilic S. elongatus. Likewise, substantial N-terminal truncations of PsbO from Spinacea oleracea by 15 or 18 residues or C-terminal deletions of 3 or 4 residues were found to interfere with an ability of the higher-plant protein to efficiently bind to PSII and reconstitute oxygen evolution activity [43,50,90,94]. These results suggest that the optimal total length of the PsbO protein is an important parameter in maintaining the functional structure that assembles into PSII. A possible cause for this phenomenon may be the neces-

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Fig. 1. Overlap of the three-dimensional homology models of PsbO in the PSII-associated form from a higher plant (Spinacea oleracea, purple), green alga (Chlamydomonas reinhardtii, green), and red alga (Porphyra yezoensis, red) with the crystallographic model of PsbO from cyanobacteria (Thermosynechococcus elongatus, cyano blue). A crystallographic model of PsbO from T. elongatus [PDB entry 3bz2] [2] was used as a template. Zoom in A, The black arrows mark the cyanobacterial loop previously denoted as the cyano-loop [28] and the loops in red algal, green algal, and higher plant PsbOs that are homologous with the cyano-loop; Zoom in B, the large flexible loop of PsbO, black dashed lines mark the putative electrostatic interaction between R151 and D224, D157 and R161, and K190 and D226 in the spinach model. The homology models were constructed using the DeepView/Swiss-PDBViewer program [see 52], available on the Internet (http://spdbv.vital-it.ch/), and the SWISS-MODEL server [137–139] (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

sity to maintain essential interactions between the N- and C-termini of the protein (95). It has been proposed that PsbO binds first to the PSII docking site, and then undergoes folding that enables the protein its functional assembly into PSII [27,50,51,96,97]. This folding process increases the protein’s content of b-sheet at the expense of random coil [51] without affecting the shape/size and domain structure of PsbO [96], the interaction between the C- and N-termini, and the hydrophobic C-terminus [2,32,33,43,49,50,95,98], as compared to the PsbO structure in solution. While the N-terminal residues of PsbO are important for the first binding step [43,91], the amino acid residues affecting the second binding step were found to be dispersed in various domains of PsbO [49,50,68,94,99]. Studies that employed site-directed mutagenesis to replace R151, R161, and Asp157 in the large flexible loop of PsbO showed that these residues have little or no effect on initial binding of PsbO to PSII, but they significantly affect the subsequent functional folding and assembly of PsbO into PSII [68,97,99], which has an adverse effect on retention of the inorganic cofactors by the OEC (see also the Section 3. Function of PsbO in PSII). A spectroscopic study that examined the contribution of aromatic residues at the PsbO C-terminus to the activity and spectral properties of PsbO by substituting Phe for W241 and Y242 revealed that the PsbO C-terminus plays a major role in functional assembly of PsbO into PSII [49,52]. This is consistent with the results of an acetylation study, where PsbO Tyr residues were modified using N-acetlylimidazole [100], and with the earlier work by Betts et al. [94] and Lydakis-Simantiris et al. [50], who showed using the C-terminal truncations of PsbO that Leu245 is essential for the protein’s assembly into PSII. Taken together, both the C-terminus and the large flexible loop of PsbO appear to be important for functional assembly of PsbO into PSII. The requirement for the intact PsbO

C-terminus in this process is consistent with the observations that this region of both solubilized and PSII-associated PsbO is buried in a hydrophobic domain [2,32–34,98], destabilization of which would likely disrupt the b-barrel structure of the protein (see Fig. 1). In contrast, the adverse effect of mutations in the large flexible loop of PsbO on PsbO–PSII interaction is more puzzling, owing to the flexibility of unordered turns and coils, which permits them to accommodate structural requirements for protein–protein interactions [39,45]. One possible explanation could perhaps be found in putative salt bridges between conserved amino acid residues of PsbO that are localized in unordered coils and turns, because these interactions require a precise positioning of the appropriate amino acid side-chains, and mutations might impose a prohibitively high energetic cost on this requirement [101]. The results from previous reports [68,97,99] would suggest that possible candidates for the intra-molecular electrostatic interactions within PsbO could be the amino acid pairs D157–R161, R151– D224 and/or K190–D226 (spinach numbering), where R151, D157, R161, and K190 are in the large flexible loop of PsbO, and D224 and D226 form a part of the small coil protruding from the PsbO b-barrel. All of these residues are fully conserved among PsbO sequences [52]. Although orientations of amino acid side-chains are not fully reliable at the 3 Å resolution of current PSII crystal structures, an attempt was made to localize these conserved residues within the backbone of PSII-associated PsbO and to assess the extent of spatial conservation of the domains carrying them. The crystallographic model of cyanobacterial PsbO from T. elongatus was used as a template (PDB entry 3bz2 [2]) to construct three homology models of PsbO in the PSII-associated form from higher plant, red and green algae. All four PsbO models are overlapped in Fig. 1, where spinach (S. oleracea) PsbO is depicted in purple, green algal (C. reinhardtii) PsbO in green, red algal (Porphyra yezoensis)

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A

B

D157

R161

C

R161

R161

D157

D157 R151

R151

R151 K190 D224

D224

D226 K190

D226 D224

K190

D226

Free in solution

Early assembly during docking of PsbO to PSII

Complete functional assembly of PsbO into PSII

Fig. 2. Schematic image of a putative mechanism for the proper folding and assembly of flexible loops of wild-type PsbO from spinach into PSII. Only the large flexible loop of PsbO (blue) and the small coil protruding from the PsbO b-barrel (green) are shown. The putative intra-molecular electrostatic interactions between the conserved amino acid pairs R151 and D224, D157 and R161, and K190 and D226 are marked by red dotted lines. The purple arrows denote the first scenario for PsbO assembly into PSII that includes panels A, B, and C; the yellow arrow stands for the second scenario that includes panels A and C only. For other details, see text (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

PsbO in red, and cyanobacterial (Thermosynechococcus elongatus) PsbO in cyano blue. The overlap in Fig. 1 reveals that after binding of PsbO to PSII, the PsbO N-terminus is accessible while its C-terminus is buried in a hydrophobic environment. This is consistent with the discovery by Seidler [69] that a His-tail introduced on the PsbO N-terminus maintains wild-type activity of the protein, but a C-terminal His-tail yields PsbO with impaired activity, and with the results of Liu et al. [102], who restored the defective psbo1 phenotype with an N-terminal His6-tagged PsbO-1 protein in transgenic A. thaliana plants. Fig. 1 also shows that the shape of the b-barrel is almost identical for all four PsbO variants, but two significant flexible domains of PsbO exhibit differences: the domain previously denoted as the cyano-loop [28] and the large flexible domain of PsbO. The cyano-loop is largest in cyanobacterial PsbO, where it could compensate for the absence of the N-terminal PsbO sequence so as to keep the protein’s length the same as that of higher plant PsbO (see discussion above and in [52]). The loop in red algal PsbO that is homologous with the cyano-loop is slightly shorter, and exhibits at least 50% spatial overlap with it. In contrast, the loop in green algal PsbO that is homologous with the cyano-loop is significantly shorter, indicating the existence of considerable divergence of red and green algal PsbOs during evolution, in agreement with [31,52,74,103,104]. As green algal PsbO is likely to have given rise to PsbO from higher plants [31,74], evolutionarily the highest form of PsbO, the loop in spinach PsbO that is homologous with the cyano-loop is shortest of all four variants, as can be expected based on the presence of the longest N-terminus in higher plant PsbO’s. As regards the large flexible loop of PsbO, it differs among PsbO species in the sequence between the residues A172 and N188 (spinach numbering), but the conserved amino acids R151, D157, R161, and K190 (spinach numbering) are located in the domains that are spatially overlapped among PsbOs, as is the coil containing D224 and D226 (Fig. 1). It is possible that these spatially overlapping domains of PsbO are important for the functional structure of the protein and that the position of these domains remained spatially conserved during evolution, because they carry conserved amino acid residues that are involved in important intra-molecular interaction(s). The results that are currently available [68,97,99] reveal the adverse effect of mutations in the large flexible loop of PsbO on functional assembly of PsbO into PSII, but they are unable to explain the nature of the defect. Therefore, a two-scenario mechanism for this phenomenon is proposed here using the schematic image in Fig. 2. The first scenario (the purple arrows; panels A ? B ? C) assumes that the flexible loops of PsbO could gain some minimal structural

organization that is stabilized by the intra-molecular electrostatic interactions that are established in the early assembly process during docking of wild-type PsbO into PSII (Fig. 2B), as opposed to the situation in solution, where no such interactions between conserved residues exist (Fig. 2A). If these putative intra-molecular salt bridges are disrupted by mutation, they cannot be easily reestablished with the substituted residues, even if these residues are very similar (for example, the D157E mutation in [68,97]), because the salt-bridge partners require the exact positioning of their sidechains [101]. As a result, the mutated large flexible loop of PsbO, in contrast to its wild-type version (Fig. 2C), cannot adopt an optimal shape that allows its proper folding needed for functional assembly of the protein into PSII. According to the second scenario (the yellow arrow; panels A ? C), which omits the stage depicted in Fig. 2B, the intra-molecular interactions between the conserved amino acid pairs are established directly from the solubilized form of PsbO (Fig. 2A) in a stepwise process during assembly of PsbO into PSII. Elimination of any of these electrostatic interactions (steps) by mutation disrupts the folding events that take place in WT PsbO (Fig. 2C); i.e. the complete and proper functional association of the protein with PSII. Currently, it is difficult to choose one scenario over the other. Hopefully, future research will provide better insights into this important process that, if not properly established, can significantly affect the efficiency of the mechanism of water oxidation.

3. Function of PsbO in PSII 3.1. PsbO and the OEC active site Despite different structural organizations of PSII in various photosynthetic organisms [31,74], it is generally assumed that the relationship between PsbO and the OEC active site and the basic principles by which PsbO increases efficiency of the water-oxidation reaction are likely to be the same in all oxygenic organisms. In the following sections, these assumptions will be used, because the majority of experiments to characterize PsbO function are based on biochemical studies on spinach PSII, while structural analyses of PsbO in its PSII-associated form come from the cyanobacterial crystal structures of PSII. Fig. 3A shows the crystallographic model of the OEC active site from T. elongatus (PDB entry 3BZ2 [2]). For purposes of clarity, extrinsic subunits (PsbO, PsbV, and PsbU) are fully visualized, but only the domains of the PSII core polypeptides (D1, D2, CP43, and CP47) are shown that reside in the

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experiments [73] showed that a PsbO-depleted PSII sample could be stabilized by lower Cl concentrations. For example, concentrations of Cl between 10 and 17 mM preserve 40–70% of the control activity after 5 h in darkness at 4 °C. The same concentrations preserve about 20% of the activity after prolonged incubation (23 h); a sample incubated under the same conditions with 100 mM Cl retains about 70% of the original activity. The stabilizing effect of Cl on PSII lacking PsbO can be remedied by rebinding of one copy of PsbO in the presence of 10 mM Cl . For example, one copy of DE18M that binds to PSII with low affinity preserves 60% of the initial oxygen evolution activity after long (23 h) dark incubation, while high affinity binding of one copy of DS13M PsbO stabilizes up to 90% of the initial activity. Rebinding of two PsbO subunits completely protects the original activity under the same incubation conditions. Quantification of Mn in these samples showed that the Mn content in PsbO-depleted PSII after 23 h of dark incubation at 4 °C with 10 mM Cl is 36% of the Mn observed in SW-PSII (a sample that contains native PsbO, but is depleted of PsbQ and PsbP). Low affinity binding of one copy of PsbO restores retention of about three quarters of initial Mn, while high affinity rebinding of one PsbO or two subunits to PSII completely stabilizes the initial Mn content [73]. These results indicate that a single copy of PsbO is sufficient to stabilize the Mn cluster in eukaryotic PSII.

Fig. 3. The crystallographic model of the OEC active site from T. elongatus (PDB entry 3bz2 [2]). (A) For purposes of clarity, the extrinsic proteins PsbO, PsbU, and PsbV are fully visualized, but only the domains of the PSII core proteins (D1, D2, CP43, and CP47) are shown that are localized in the proximity of the OEC active site. The MnCa2+ cluster and the Cl cofactor are marked in red and black, respectively. The figure shows that the large flexible loop of PsbO folds/assembles near the OEC active site. (B) A detailed view reveals the position of Cl in proximity of the PsbOD158 and PsbO-R162 residues that might form a salt bridge, which would facilitate an optimal assembly of the large flexible loop of PsbO and enable efficient retention of Cl in the OEC [68,97,99].

vicinity of the OEC active site. As can be seen in Fig. 3A, the N-terminus, C-terminus, and b-barrel of PsbO are quite distant from the MnCa cluster and the Cl cofactor, while the large flexible loop of PsbO folds and assembles near them. Thus, if there are PsbO residues that are essential for Mn redox reactions, it would be most likely those that reside in the large flexible loop of the protein. So far, this loop has been shown to carry amino acid residues that are important for a proper functioning of the Cl cofactor in the OEC active site (see the Section 3.2.3). 3.2. The role of PsbO in relation to the inorganic cofactors in the OEC 3.2.1. PsbO and manganese Manganese-stabilizing protein was the name originally given to PsbO, because it promotes retention of the Mn cluster in the presence of physiological concentrations of Ca2+ and Cl in the OEC. Under these conditions, removal of PsbO destabilizes the Mn cluster and two of four Mn are reduced, and eventually lost [105,106]. In PsbO-depleted PSII, it was originally observed that high Cl concentrations can stabilize the Mn cluster in the dark, although the rate of oxygen evolution is significantly decreased [16,18]. Later

3.2.2. PsbO and Ca2+ A recent review revisits and discusses the Ca2+ site in PSII [107]; here, the focus will be on the Ca2+ site exclusively in relation to PsbO, a topic that has given rise to some contradictory conclusions. The cyanobacterial crystal structures of PSII indicate that PsbO does not donate ligands to Ca2+ [2,32,33]. There is nevertheless a considerable literature on the relationship between PsbO and Ca2+ binding to PSII, some of which precedes the appearance of the structural information. For example, it was proposed that PsbO might be required for a normal association of Ca2+ with PSII, because PsbO decreases the requirement for added Ca2+ in PSII by a factor of 2 [18]. The amino acid sequence of PsbO contains a putative calcium-binding site [108,109] and some studies suggested that PsbO is a Ca2+-binding protein [110–112]. These results have to be taken with caution, however, because PsbO is acidic (pI 5.2), and its negative charges under physiological conditions (pH 6 or 7) could bind divalent metal ions nonspecifically. It has been shown that PsbO might bind Ca2+ [113], and there is a proposal that it is involved, together with the Mn cluster, in a creation of the Ca2+ binding site [114]. Other investigators have questioned any direct role of PsbO in Ca2+ binding [115]. An FTIR examination of spinach PsbO yielded results that were interpreted to indicate that Ca2+ induces conformational changes in PsbO structure that might facilitate its binding to PSII [116]. However, similar experiments on PsbO from T. elongatus revealed no Ca2+-modulated structural changes [117], which were interpreted to indicate that Ca2+ is not bound to PsbO before it assembles into PSII, at least in cyanobacteria. This led to a hypothesis that the Ca2+ binding site might be created upon binding of PsbO to PSII intrinsic subunits. Typical calcium-binding proteins exhibit higher binding affinities for Ca2+ [118] than does PsbO [111]. Recent steady-state assays showed that the Ca2+ KM is in the same range (0.9  10 4– 2.2  10 4 M) when PsbO-depleted PSII is reconstituted with various recombinant spinach proteins (Table 2), such as the N-terminal truncated mutants that affect PsbO stoichiometry in PSII or the mutants that substitute R151, R161 or D157 in the larger flexible loop of PsbO with some other residue. These results indicate that none of these mutations affect Ca2+ retention in PSII under steady-state illumination [68,73,99]. An investigation of the effect of PsbO on the ability of Ca2+ to protect the Mn cluster against reductive inhibition [119,120] revealed that 10 mM Ca2+ added to PSII samples protected Mn against the reductant independent of the

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Table 2 KM values for Ca2+ and Cl calculated from activity assays carried out with varying Ca2+ and Cl concentrations at pH 6.

a b c d e

Sample

Cl KM (mM)

Ca2+ KM (mM

SW-PSII UW-PSII UW-PSII + 1 PsbOa weakly bound UW-PSII + 1 PsbOb UW-PSII + 2 PsbOsc UW-PSII + R161G PsbOd UW-PSII + R151G PsbOd UW-PSII + R151D PsbOd UW-PSII + D157E PsbOe UW-PSII + D157N PsbOe UW-PSII + D157K PsbOe

0.9 3.9 2.2–2.5 1.5–1.6 1.0–1.3 1.5 2.6 2.2 1.5 1.6 1.6

0.15 0.13 0.10–0.12 0.09–0.15 0.09–0.15 0.22 0.22 0.17 0.10 0.13 0.11

DT15M or DE18M PsbO [73]. DT7M or DS13M PsbO [73]. WT or DR5M or DG3M PsbO [73]. From [99]. From [68].

presence of PsbO. Experiments are needed to determine whether this is also the case at significantly lower Ca2+ concentrations. The recent work by Bricker and Frankel showed that the psbo1 mutant from A. thaliana, which lacks PsbO-1, but not the PsbO-2 protein, cannot efficiently maintain Ca2+ in the OEC [121]. This would be consistent with the hypothesis of Loll et al. [117] that PsbO binding to PSII facilitates formation of the Ca2+ binding site. It is apparent from this discussion that more research is needed to clarify the role of PsbO with regard to the structure and function of the Ca2+ site in the OEC. It should be emphasized again that the current evidence supports the view that PsbO is not a Ca2+ binding protein per se, but the biochemical experiments on the requirement for PsbO for optimal Ca2+ binding would suggest that the protein is required to promote Ca2+ retention, perhaps by preventing release of the metal from PSII [122], by participating in formation of the functional Ca2+ binding site during biogenesis of PSII [114], or some combination of both of these hypotheses.

3.2.3. PsbO and Cl A detailed view in Fig. 3B reveals that the D158 and R162 residues in cyanobacterial PsbO, homologous to D157 and R161 in spinach PsbO, are in proximity to the inorganic cofactors in the OEC (D158 and Cl are at a distance of 14 Å, R162 and Cl are 17 Å distant, D158 and Ca2+ are 22 Å distant, and R162 and Ca2+ are 26 Å distant according to the PSII crystal structure by

Guskov et al. [2]). Both PsbO-Asp157 and PsbO-Arg161 from spinach were subjected to mutagenesis in three recent studies [68,97,99]. The results in [99] showed that R161G PsbO, R151G PsbO, and R151D PsbO exhibit lower PSII binding affinity and reconstitute only 40% or 20% of control activity. This was in agreement with the work by Motoki et al. [88] who reported that the mutations in homologous residues R152 or R162 of PsbO from thermophilic S. elongatus exhibit significantly impaired function [88]. More extensive assays on the Arg mutants of spinach PsbO revealed that all of the mutated proteins are defective in efficient retention of Cl (Table 2), suggesting that Arg151 and Arg161 play a role in functional assembly of PsbO into PSII (as mentioned above). In a study where PsbO-Asp157 was replaced with Asn, Glu, or Lys [68], the D157E, D157N, and D157K PsbO’s were found to be the first mutated proteins from spinach that exhibit wildtype PSII binding combined with low O2-evolution activity (30% of controls). All mutants were also shown to be defective in efficient retention of Cl (Table 2). Taken together, existing data indicate that both positively (Arg) and negatively (Asp) charged amino acid residues in the large flexible loop of PsbO are involved in efficient retention of Cl in the OEC; the data currently available would suggest that rebound mutated PsbO’s are unable to trap Cl effectively during steady state turnovers of the OEC, specifically in the higher S-states where Cl is rapidly lost [123]. The PsbO–Cl relationship was further characterized in a study that explored the effect of PsbO stoichiometry on the inorganic cofactors in the OEC active site. This work revealed that each copy of PsbO in eukaryotic PSII has a unique function in relation to Cl ; the first copy enhances Cl retention, while the second copy functions to optimize it [73] (see also Table 2). This is consistent with earlier results of Miyao and Murata [16,124], who showed that rebinding of PsbO decreases the optimum Cl concentration for oxygen evolution activity. Similar conclusions were also reported by Bricker in [18]. The C-terminal W241F mutation in spinach PsbO, which induces additional thermostability in the protein [49], was also found to affect efficient retention of Cl [52]. Given the distance between the PsbO C-terminus and the OEC active site (Fig. 3A), optimal retention of Cl in the OEC appears to require that PsbO is able to exploit its structural flexibility in solution in such a way that it undergoes overall refolding and assembles into PSII [52].

3.2.4. Summary The schematic model in Fig. 4 summarizes PsbO function in relation to all inorganic cofactors and to the action of OH and

Fig. 4. Schematic model of PsbO function in relation to the Mn, Ca2+, and Cl cofactors and to the action of OH and small Mn reductants in the eukaryotic OEC. For purposes of simplicity, the smaller PSII polypeptides are omitted and only the PSII intrinsic proteins D1, D2, CP43, and CP47, and the extrinsic PsbO polypeptide are shown. On the left, the OEC active site with two PsbO subunits; in the middle, the OEC active site with one PsbO subunit maintaining the intact Mn cluster; on the right, the OEC active site with no extrinsic proteins and the disassembled Mn cluster. For other details, see text.

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small Mn reductants in eukaryotic OEC. Binding of two PsbO subunits in eukaryotic PSII (Fig. 4, left) assures a proper ligation and efficient retention of Ca2+ and Cl , leading to a high rate of oxygen evolution (which requires two PsbO’s per PSII in eukaryotes) and to a full protection of the intact Mn cluster against OH and small reductants, such as dimethylhydroxylamine (DMHA) [68,73,120]. In contrast, the loss of one PsbO subunit from eukaryotic PSII (Fig. 4, middle) decreases Cl retention in the OEC [73]. This likely increases a competitiveness of OH for the PSII binding site and its chance for an inhibitory interaction with Mn. However, with one PsbO subunit present in PSII, the Cl concentration is still sufficient to stabilize the intact Mn cluster; PsbO was found to stabilize the cluster through Cl retention [16,18,73]. With one PsbO in the OEC, Ca2+ at the concentration of 10 mM is bound near the Mn cluster, and is able to protect the cluster against reductant attack and inhibition [73]. Fig. 4, right shows that without PsbO, Ca2+ (at 10 mM) can protect the Mn cluster against reductive inhibition [73,119,120], but it might not be properly ligated with respect to the Mn atoms, or a proper binding site might not be created [117,121]. In the absence of PsbO, Cl is not efficiently retained in the OEC [16,73], thus, OH can outcompete Cl for the PSII binding site [125], and inhibit the Mn cluster [68]. As a result, the Mn cluster dissociates and the Mn2+ ions are released from the OEC active site [16,73]. This indicates that PsbO might have a dual role in the OEC, i.e. retention of Cl and a function as a barrier to protect against OH . 3.3. The effect of PsbO on the S-states and Y
Z A number of studies suggest an intimate relationship of PsbO with the Mn cluster [16,18,73,106], which is in agreement with the findings that the PsbO protein affects cycling of the Mn cluster through the S-states. Examination of the S2-state multiline EPR signal showed a decrease in signal intensity due to PsbO extraction from PSII [126]. Flash oxygen yield measurements conducted by Miyao and Murata [127] revealed that removal of PsbO from PSII causes a higher dark stability of the S2 and S3 states and that kinetics for O2 release after the third flash is retarded, indicating the slower S3 to S0 transition. This was consistent with a thermoluminescence study by Ono and Inoue who observed that the S3 ? [S4] ? S0 transition was inhibited after PsbO depletion [128]. In agreement with biochemical extraction of PsbO from PSII, the cyanobacterial DpsbO mutants also exhibited the slower S3 ? [S4] ? S0 transition and an increased stabilization of the S2 and S3 states [129,130]. Biochemical and biophysical studies with the spinach PsbO protein showed that aspartate and/or glutamate residues of PsbO deprotonate during the S1 ? S2 transition [131], and that the structure of PsbO is changed upon reduction of the Mn cluster with NH2OH [132]. A recent work [97] that employed site-directed mutants of spinach PsbO showed that Asn, Glu or Lys mutations in PsbO-Asp157 affect the recombination of Q A with the OEC, and that oxygen yield on the first flash is increased in mutant samples, indicating a defect in ability of PSII reconstituted with PsbO-Asp157 mutants to decay back to the S1 state in darkness. Lastly, evidence has been presented to show that reduction of Y
Z is slowed from 1.2 to about 6 ms in PSII lacking PsbO [133]. 4. Conclusions As both Cl and Ca2+ are required for the S-state transitions in the OEC [107,123,134,135], it could be hypothesized that PsbO might affect stability and transition of the S-states through its effect on the inorganic cofactors in the OEC. Probing this hypothesis deserves more in depth investigation. In addition to what has already been mentioned in this minireview, PsbO was also suggested

187

to be a part of the putative channels that link the OEC active site with the lumen; specifically, PsbO-Asp157 was proposed to be involved in a proton transfer network [2,28,136]. Recent work on D157 mutants using chemical rescue by proton acceptors were unsuccessful in further increasing activity that was partially restored by reconstitution of Asp157 mutants into PsbO-depleted PSII. This result was interpreted to indicate that PsbO-D157 is not involved in the proton transfer network [68,97]. However, this finding does not exclude a possibility that some other PsbO residue participates in H+-transporting channels near the OEC. It is also possible that PsbO residues might be involved in PSII redox reactions or they might play an optimizing role in maximizing oxygen evolution activity of PSII. Despite extensive research that has produced a significant amount of data on PsbO structure and function, this interesting protein still has a potential for new discoveries that will lead to a complete description of its role in PSII. 5. Abbreviations DMHA EDC EPR FTIR OEC PS PsbO PsbV PsbU QA(B) NHS Y
Z

dimethylhydroxylamine 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide electron paramagnetic resonance Fourier transfer infrared oxygen-evolving complex photosystem the manganese-stabilizing protein cytochrome c550 12 kDa extrinsic protein primary (secondary) quinone acceptor of PSII N-hydroxysucinimide radical of redox-active tyrosine

Acknowledgement H.P. and C.F.Y. acknowledge support from the National Science Foundation (MCB-0716541). References [1] N. Nelson, C.F. Yocum, Structure and function of photosystems I and II, Annu. Rev. Plant Biol. 57 (2006) 521–565. [2] A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution and the role of quinones, lipids, channels, and chloride, Nature Struct. Mol. Biol. 16 (2009) 334–342. [3] R.J. Debus, Protein ligation of the photosynthetic oxygen-evolving center, Coord. Chem. Rev. 252 (2008) 244–258. [4] I.L. McConnell, Substrate water binding and oxidation in photosystem II, Photosynth. Res. 98 (2008) 261–276. [5] T.J. Wydrzynski, Water splitting by photosystem II – Where do we go from here?, Photosynth Res. 98 (2008) 43–51. [6] C.F. Yocum, The calcium and chloride requirements of the O2 evolving complex, Coord. Chem. Rev. 252 (2008) 296–305. [7] F.E. Callahan, G.M. Cheniae, Studies on the photoactivation of the wateroxidizing enzyme. 1. Processes limiting photoactivation in hydroxylamineextracted leaf segments, Plant Physiol. 79 (1985) 777–786. [8] F.E. Callahan, D.W. Becker, G.M. Cheniae, Studies on the photoactivation of the water-oxidizing enzyme. 2. Characterization of weak light photoinhibition of PSII and its light-induced recovery, Plant Physiol. 82 (1986) 261–269. [9] N. Tamura, Y. Inoue, G.M. Cheniae, Photoactivation of the water-oxidizing complex in photosystem II membranes depleted of Mn, Ca and extrinsic proteins. II. Studies on the functions of Ca2+, Biochim. Biophys. Acta 976 (1989) 173–181. [10] C. Chen, J. Kazimir, G.M. Cheniae, Calcium modulates the photoassembly of photosystem II (Mn)4-clusters by preventing ligation of nonfunctional highvalency states of manganese, Biochemistry 34 (1995) 13511–13526. [11] N. Tamura, G.M. Cheniae, Photoactivation of the water-oxidizing complex in photosystem II membranes depleted of Mn and extrinsic proteins. 1. Biochemical and kinetic characterization, Biochim, Biophys. Acta 890 (1987) 179–194. [12] W.G. Gregor, R.M. Cinco, H. Yu, V.K. Yachandra, R.D. Britt, Influence of the 33 kDa manganese stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II, Biochemistry 44 (2005) 8817–8825.

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